Lactiplantibacillus plantarum 299v (LP299V®): three decades of research.

This review aims to provide a comprehensive overview of the in vitro, animal, and clinical studies with the bacterial strain Lactiplantibacillus plantarum 299v (L. plantarum 299v; formerly named Lactobacillus plantarum 299v) published up until June 30, 2020. L. plantarum 299v is the most documented L. plantarum strain in the world, described in over 170 scientific publications out of which more than 60 are human clinical studies. The genome sequence of L. plantarum 299v has been determined and is available in the public domain (GenBank Accession number: NZ_LEAV01000004). The probiotic strain L. plantarum 299v was isolated from healthy human intestinal mucosa three decades ago by scientists at Lund University, Sweden. Thirty years later, a wealth of data coming from in vitro, animal, and clinical studies exist, showing benefits primarily for gastrointestinal health, such as reduced flatulence and abdominal pain in patients with irritable bowel syndrome (IBS). Moreover, several clinical studies have shown positive effects of L. plantarum 299v on iron absorption and more recently also on iron status. L. plantarum 299v is safe for human consumption and does not confer antibiotic resistance. It survives the harsh conditions of the human gastrointestinal tract, adheres to mannose residues on the intestinal epithelial cells and has in some cases been re-isolated more than ten days after administration ceased. Besides studying health benefits, research groups around the globe have investigated L. plantarum 299v in a range of applications and processes. L. plantarum 299v is used in many different food applications as well as in various dietary supplements. In a freeze-dried format, L. plantarum 299v is robust and stable at room temperature, enabling long shelf-lives of consumer healthcare products such as capsules, tablets, or powder sachets. The strain is patent protected for a wide range of indications and applications worldwide as well as trademarked as LP299V®.

Neuronal metabolic rewiring promotes resilience to neurodegeneration caused by mitochondrial dysfunction.

Neurodegeneration in mitochondrial disorders is considered irreversible because of limited metabolic plasticity in neurons, yet the cell-autonomous implications of mitochondrial dysfunction for neuronal metabolism in vivo are poorly understood. Here, we profiled the cell-specific proteome of Purkinje neurons undergoing progressive OXPHOS deficiency caused by disrupted mitochondrial fusion dynamics. We found that mitochondrial dysfunction triggers a profound rewiring of the proteomic landscape, culminating in the sequential activation of precise metabolic programs preceding cell death. Unexpectedly, we identified a marked induction of pyruvate carboxylase (PCx) and other anaplerotic enzymes involved in replenishing tricarboxylic acid cycle intermediates. Suppression of PCx aggravated oxidative stress and neurodegeneration, showing that anaplerosis is protective in OXPHOS-deficient neurons. Restoration of mitochondrial fusion in end-stage degenerating neurons fully reversed these metabolic hallmarks, thereby preventing cell death. Our findings identify a previously unappreciated pathway conferring resilience to mitochondrial dysfunction and show that neurodegeneration can be reversed even at advanced disease stages.

Modulation of mtDNA copy number ameliorates the pathological consequences of a heteroplasmic mtDNA mutation in the mouse.

Heteroplasmic mtDNA mutations typically act in a recessive way and cause mitochondrial disease only if present above a certain threshold level. We have experimentally investigated to what extent the absolute levels of wild-type (WT) mtDNA influence disease manifestations by manipulating TFAM levels in mice with a heteroplasmic mtDNA mutation in the tRNAAla gene. Increase of total mtDNA levels ameliorated pathology in multiple tissues, although the levels of heteroplasmy remained the same. A reduction in mtDNA levels worsened the phenotype in postmitotic tissues, such as heart, whereas there was an unexpected beneficial effect in rapidly proliferating tissues, such as colon, because of enhanced clonal expansion and selective elimination of mutated mtDNA. The absolute levels of WT mtDNA are thus an important determinant of the pathological manifestations, suggesting that pharmacological or gene therapy approaches to selectively increase mtDNA copy number provide a potential treatment strategy for human mtDNA mutation disease.

Heparin-binding protein in ventilator-induced lung injury.

BACKGROUND: Although mechanical ventilation is often lifesaving, it can also cause injury to the lungs. The lung injury is caused by not only high pressure and mechanical forces but also by inflammatory processes that are not fully understood. Heparin-binding protein (HBP), released by activated granulocytes, has been indicated as a possible mediator of increased vascular permeability in the lung injury associated with trauma and sepsis. We investigated if HBP levels were increased in the bronchoalveolar lavage fluid (BALF) or plasma in a pig model of ventilator-induced lung injury (VILI). We also investigated if HBP was present in BALF from healthy volunteers and in intubated patients in the intensive care unit (ICU). METHODS: Anaesthetized pigs were randomized to receive ventilation with either tidal volumes of 8 ml/kg (controls, n = 6) or 20 ml/kg (VILI group, n = 6). Plasma and BALF samples were taken at 0, 1, 2, 4, and 6 h. In humans, HBP levels in BALF were sampled from 16 healthy volunteers and from 10 intubated patients being cared for in the ICU. RESULTS: Plasma levels of HBP did not differ between pigs in the control and VILI groups. The median HBP levels in BALF were higher in the VILI group after 6 h of ventilation compared to those in the controls (1144 ng/ml (IQR 359-1636 ng/ml) versus 89 ng/ml (IQR 33-191 ng/ml) ng/ml, respectively, p = 0.02). The median HBP level in BALF from healthy volunteers was 0.90 ng/ml (IQR 0.79-1.01 ng/ml) as compared to 1959 ng/ml (IQR 612-3306 ng/ml) from intubated ICU patients (p < 0.001). CONCLUSIONS: In a model of VILI in pigs, levels of HBP in BALF increased over time compared to controls, while plasma levels did not differ between the two groups. HBP in BALF was high in intubated ICU patients in spite of the seemingly non-harmful ventilation, suggesting that inflammation from other causes might increase HBP levels.

Percutaneously inserted long-term central venous catheters in pigs of different sizes.

Pigs are used for long-term biomedical experiments requiring repeated injections, infusions and collections of blood samples. Thus, it is necessary for vascular catheters to be indwelling to avoid undue stress to the animals and the use of restraints. We propose a refined model of percutaneous insertion of long-term central venous catheters to minimize the surgical trauma and postoperative complications associated with catheter insertion. Different sizes of needles (18 Ga versus 21 Ga) for initial puncture of the veins were compared. In conventional pigs weighing less than 30 kg, catheter insertion may be facilitated by using a microintroducer set with a 21 Ga needle. In pigs weighing 50 kg, a standard 18 Ga needle may be preferable.

The role of mitochondrial DNA mutations and free radicals in disease and ageing.

Considerable efforts have been made to understand the role of oxidative stress in age-related diseases and ageing. The mitochondrial free radical theory of ageing, which proposes that damage to mitochondrial DNA (mtDNA) and other macromolecules caused by the production of reactive oxygen species (ROS) during cellular respiration drives ageing, has for a long time been the central hypothesis in the field. However, in contrast with this theory, evidence from an increasing number of experimental studies has suggested that mtDNA mutations may be generated by replication errors rather than by accumulated oxidative damage. Furthermore, interventions to modulate ROS levels in humans and animal models have not produced consistent results in terms of delaying disease progression and extending lifespan. A number of recent experimental findings strongly question the mitochondrial free radical theory of ageing, leading to the emergence of new theories of how age-associated mitochondrial dysfunction may lead to ageing. These new hypotheses are mainly based on the underlying notion that, despite their deleterious role, ROS are essential signalling molecules that mediate stress responses in general and the stress response to age-dependent damage in particular. This novel view of ROS roles has a clear impact on the interpretation of studies in which antioxidants have been used to treat human age-related diseases commonly linked to oxidative stress.

MitoPark mice mirror the slow progression of key symptoms and L-DOPA response in Parkinson's disease.

The MitoPark mouse, in which the mitochondrial transcription factor Tfam is selectively removed in midbrain dopamine (DA) neurons, is a genetic model for Parkinson's disease (PD) that replicates the slow and progressive development of key symptoms. To further validate this model, we have extended both behavioral and biochemical analyses in these animals. We found that vertical movements decline earlier and faster than horizontal movements, possibly modeling the early occurrence of axial, postural instability in PD. L-DOPA induces different locomotor responses depending on the age: in young MitoPark mice the L-DOPA-induced motor activation is small; middle-aged MitoPark mice respond in a dose-dependent manner to L-DOPA, whereas aged MitoPark mice display a double-peaked locomotor response to a high dose of L-DOPA that includes an intermittent period of very low motor activity, similar to the 'on-off' phenomenon in PD. To correlate behavior with biochemical data, we analyzed monoamine levels in three different brain areas that are highly innervated by the DA system: striatum, anterior cortex and olfactory bulb. DA levels declined earlier and faster in striatum than in cortex; only at the latest time-point analyzed, DA levels were found to be significantly lower than control levels in the olfactory bulb. Interestingly, the ratio between homovanillic acid (HVA) and DA differed between regions over time. In striatum and olfactory bulb, the ratio increased steeply indicating increased DA turnover. In contrast, the ratio decreased over time in cortex, revealing important differences between DA cells in substantia nigra and the ventral tegmental area.

Analysis and quantification of parabens in cosmetic products by utilizing hollow fibre-supported liquid membrane and high performance liquid chromatography with ultraviolet detection.

A simple and direct method based on hollow fibre-supported liquid membrane (HFSLM) extraction and liquid chromatography equipped with a UV detector was developed for analysis and quantification of parabens in cosmetic products. The parabens analysed included methyl, ethyl, propyl, isobutyl and butyl paraben. The HFSLM extraction was carried out by employing di-n-hexyl ether as organic liquid that was immobilized in the hollow fibre membrane. The HFSLM extraction is simple, cheap, minimizes the use of solvents and uses disposable material. In an investigation of 11 paraben-containing cosmetic products, the levels of parabens (sum of all parabens in a product) ranged from 0.43% to 0.79% (w/w) for skin care products, 0.07-0.44% for hair fixing gels and 0.30-0.52% for soap solutions. The levels of individual parabens in individual cosmetic products ranged between 0.03% and 0.42% w/w for skin care products, 0.07% and 0.26% w/w for hair fixing gels and between 0.11% and 0.34% w/w for soap solutions. Parabens were found in the highest concentrations in skin care products followed by soap solutions and the least amounts were found in hair fixing gels. Of the paraben-containing products tested, all of them contained methyl paraben and about 90% contained propyl paraben in addition to methyl paraben. One product contained all the parabens analysed.

Mitochondrial dysfunction as a cause of ageing.

Mitochondrial dysfunction is heavily implicated in the ageing process. Increasing age in mammals correlates with accumulation of somatic mitochondrial DNA (mtDNA) mutations and decline in respiratory chain function. The age-associated respiratory chain deficiency is typically unevenly distributed and affects only a subset of cells in various human tissues, such as heart, skeletal muscle, colonic crypts and neurons. Studies of mtDNA mutator mice has shown that increased levels of somatic mtDNA mutations directly can cause a variety of ageing phenotypes, such as osteoporosis, hair loss, greying of the hair, weight reduction and decreased fertility. Respiratory-chain-deficient cells are apoptosis prone and increased cell loss is therefore likely an important consequence of age-associated mitochondrial dysfunction. There is a tendency to automatically link mitochondrial dysfunction to increased generation of reactive oxygen species (ROS), however, the experimental support for this concept is rather weak. In fact, respiratory-chain-deficient mice with tissue-specific mtDNA depletion or massive increase of point mutations in mtDNA typically have minor or no increase of oxidative stress. Mitochondrial dysfunction is clearly involved in the human ageing process, but its relative importance for mammalian ageing remains to be established.

The orphan receptor GPR55 is a novel cannabinoid receptor.

BACKGROUND: The endocannabinoid system functions through two well characterized receptor systems, the CB1 and CB2 receptors. Work by a number of groups in recent years has provided evidence that the system is more complicated and additional receptor types should exist to explain ligand activity in a number of physiological processes. EXPERIMENTAL APPROACH: Cells transfected with the human cDNA for GPR55 were tested for their ability to bind and to mediate GTPgammaS binding by cannabinoid ligands. Using an antibody and peptide blocking approach, the nature of the G-protein coupling was determined and further demonstrated by measuring activity of downstream signalling pathways. KEY RESULTS: We demonstrate that GPR55 binds to and is activated by the cannabinoid ligand CP55940. In addition endocannabinoids including anandamide and virodhamine activate GTPgammaS binding via GPR55 with nM potencies. Ligands such as cannabidiol and abnormal cannabidiol which exhibit no CB1 or CB2 activity and are believed to function at a novel cannabinoid receptor, also showed activity at GPR55. GPR55 couples to Galpha13 and can mediate activation of rhoA, cdc42 and rac1. CONCLUSIONS: These data suggest that GPR55 is a novel cannabinoid receptor, and its ligand profile with respect to CB1 and CB2 described here will permit delineation of its physiological function(s).

Germline E-cadherin mutations in familial lobular breast cancer.

BACKGROUND: The cell surface glycoprotein E-cadherin (CDH1) is a key regulator of adhesive properties in epithelial cells. Germline mutations in CDH1 are well established as the defects underlying hereditary diffuse gastric cancer (HDGC) syndrome, and an increased risk of lobular breast cancer (LBC) has been described in HDGC kindreds. However, germline CDH1 mutations have not been described in patients with LBC in non-HDGC families. This study aimed to investigate the frequency of germline CDH1 mutations in patients with LBC with early onset disease or family histories of breast cancer without DGC. METHODS: Germline DNA was analysed in 23 women with invasive lobular or mixed ductal and lobular breast cancers who had at least one close relative with breast cancer or had themselves been diagnosed before the age of 45 years, had tested negative for a germline BRCA1 or BRCA2 mutation, and reported no personal or family history of diffuse gastric cancer. The full coding sequence of CDH1 including splice junctions was amplified using PCR and screened for mutations using DHPLC and sequencing. RESULTS: A novel germline CDH1 truncating mutation in the extracellular portion of the protein (517insA) was identified in one woman who had LBC at the age of 42 years and a first degree relative with invasive LBC. CONCLUSIONS: Germline CDH1 mutations can be associated with invasive LBC in the absence of diffuse gastric cancer. The finding, if confirmed, may have implications for management of individuals at risk for this breast cancer subtype. Clarification of the cancer risks in the syndrome is essential.

Adenosine is not a direct GHSR agonist--artificial cross-talk between GHSR and adenosine receptor pathways.

AIM: To assess if adenosine is a direct growth hormone secretagogue receptor (GHSR) agonist by investigating the mechanism behind adenosine induced calcium release in human embryonic kidney 293s (HEK) cells expressing GHSR. METHODS: Calcium mobilization, cyclic adenosine monophosphate (cAMP) and IP(3) experiments were performed using HEK cells stably expressing GHSR and/or adenosine A(2B) receptor (A(2B)R). RESULTS: Adenosine has been widely reported as a GHSR agonist. In our hands, adenosine and forskolin stimulated calcium release from IP(3) controlled stores in HEK-GHSR cells but not in non-transfected HEK cells. This release was not accompanied by increased IP(3) levels. The calcium release was both cholera toxin and U73122 sensitive, indicating the involvement of both Galpha(s)/adenylyl cyclase and Galpha(q/11)/phospholipase C pathways. Importantly, the GHSR inverse agonist [D-Arg(1) D-Phe(5) D-Trp(7,9) Leu(11)]-Substance P (SP-analogue) blocked the adenosine stimulated calcium release, demonstrating that GHSR is involved. Assessment of the GHSR-dependent calcium release using adenosine receptor agonists and antagonists resulted in a rank order of potencies resembling the profile of A(2B)R. A(2B)R over-expression in HEK-GHSR cells enhanced potency and efficacy of the adenosine induced calcium release without increasing IP(3) production. Moreover, A(2B)R over-expression in HEK cells potentiated NECA-induced cAMP production. However, GHSR expression had no effect on intracellular cAMP production. CONCLUSION: In HEK-GHSR cells adenosine activates endogenously expressed A(2B)R resulting in calcium mobilization. We hypothesize that the responsible mechanism is cAMP-dependent sensitization of IP(3) receptors for the high basal level of IP(3) caused by GHSR constitutive activity. Altogether, our results demonstrate that adenosine is not a direct GHSR agonist.

Defective assembly of the respiratory chain.

UNLABELLED: A functional respiratory chain is dependent on protein components encoded by both mtDNA and nuclear DNA. Isolated cytochrome c oxidase (COX) deficiency is often caused by mutations in nuclear genes regulating the assembly of the 13 protein subunits of this complex. The accompanying paper by Zeman and co-workers reports that mutations in SCO2 are common in infantile COX deficiency and are associated with a very poor prognosis. CONCLUSION: Molecular diagnosis is often feasible in patients with COX deficiency and particular attention should be paid to mutations in COX assembly genes.

Animal models for respiratory chain disease.

Elucidation of the pathogenesis in respiratory chain diseases is of great importance for developing specific treatments. The limitations inherent to the use of patient material make studies of human tissues often difficult and the mouse has therefore emerged as a suitable model organism for studies of respiratory chain diseases. In this review, we present an overview of the field and discuss in depth a few examples of animal models reproducing pathology of human disease with primary and secondary respiratory chain involvement.

Proliferation of primitive myeloid progenitors can be reversibly induced by HOXA10.

Recent studies show that several Hox transcription factors are important for regulation of proliferation and differentiation in hematopoiesis. Among these is H0XA10, which is selectively expressed at high levels in the most primitive subpopulation of human CD34(+) bone marrow cells. When overexpressed, H0XA10 increases the proliferation of early progenitor cells and can lead to the development of myeloid leukemia. To study the effects of H0XA10 on primitive hematopoietic progenitors in more detail, transgenic mice were generated with regulatable H0XA10 expression. The transgenic mouse model, referred to as tetO-HOXA10, contains the H0XA10 gene controlled by a tetracycline-responsive element and a minimal promoter. Thus, the expression of H0XA10 is inducible and reversible depending on the absence or presence of tetracycline or its analog, doxycycline. A retroviral vector containing the tetracycline transactivator gene (tTA) was used to induce expression of the H0XA10 gene in bone marrow cells from the transgenic mice. Reverse transcription-polymerase chain reaction analysis confirmed regulatable H0XA10 expression in several transgenic lines. H0XA10 induction led to the formation of hematopoietic colonies containing blastlike cells and megakaryocytes. Moreover, the induction of H0XA10 resulted in significant proliferative advantage of primitive hematopoietic progenitors (spleen colony-forming units [CFU-S(12)]), which was reversible on withdrawal of induction. Activation of H0XA10 expression in tet0-H0XA10 mice will therefore govern proliferation of primitive myeloid progenitors in a regulated fashion. This novel animal model can be used to identify the target genes of HOXA10 and better clarify the specific role of HOXA10 in normal and malignant hematopoiesis.

Liquid chromatographic determination of total and beta-N-oxalyl-L-alpha,beta-diaminopropionic acid in lathyrus sativus seeds using both refractive index and bioelectrochemical detection.

A further improved chromatographic method for the simultaneous determination of the total amount of ODAP, selectively the amount of its neurotoxic form, beta-ODAP, and free L-glutamate in raw Lathyrus sativus (grass pea) seed samples is described using post-column refractive index in combination with bioelectrochemical detection. The biosensor is based on crosslinking horseradish peroxidase (HRP) and an Os-containing mediating polymer with poly(ethyleneglycol)(400) diglycidyl ether (PEGDGE), forming an inner hydrogel layer and then immobilising L-glutamate oxidase (GlOx) as an outer layer on top of a graphite electrode. Addition of polyethylenimine (PEI) to the hydrogel is believed to have sensitivity and stability enhancing effect on the biosensor. The double-layer approach in the biosensor construction avoided direct electrical wiring of GlOx and resulted in a higher sensitivity of 4.6 mA/M cm2 with respect to beta-ODAP and a wider linear range (1-250 microM) for both L-glutamate and beta-ODAP when compared with a single-layer approach where GlOx, HRP, and Os-polymer are crosslinked together. The limit of detection for the chromatographic-biosensor system was found to be 2 microM with respect to beta-ODAP and 0.7 microM with respect to L-glutamate. The refractive index detection on-line with the biosensor enabled full control of the chromatographic system for the determination of the total amount of ODAP, selectively the amount of beta-ODAP and L-glutamate. Ten grass pea samples have been collected from Lathyrism prone areas of Ethiopia to test the applicability of the presently developed analytical system for real sample analysis. The toxin levels of grass pea collections were determined in an aqueous extracts and ranged from 0.52 to 0.76%, dry mass basis. Comparison of results of an established spectrophotometric assay and that of the present system has shown an extraordinary degree of agreement as revealed by parallel "t" test (90% confidence limit). The present system has operational stability of more than 50 h. Analysis time per sample is 10 min after extraction for 90 min.

Late-onset corticohippocampal neurodepletion attributable to catastrophic failure of oxidative phosphorylation in MILON mice.

We generated mitochondrial late-onset neurodegeneration (MILON) mice with postnatal disruption of oxidative phosphorylation in forebrain neurons. They develop normally and display no overt behavioral disturbances or histological changes during the first 5 months of life. The MILON mice display reduced levels of mitochondrial DNA and mitochondrial RNA from 2 and 4 months of age, respectively, and severely respiratory chain-deficient neurons from 4 months of age. Surprisingly, these respiratory chain-deficient neurons are viable for at least 1 month without showing signs of neurodegeneration or major induction of defenses against oxidative stress. Prolonged neuronal respiratory chain deficiency is thus required for the induction of neurodegeneration. Before developing neurological symptoms, MILON mice show increased vulnerability to excitotoxic stress. We observed a markedly enhanced sensitivity to excitotoxic challenge, manifest as an abundance of terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling (TUNEL) reactive cells after kainic acid injection, in 4-month-old MILON mice, showing that respiratory chain-deficient neurons are more vulnerable to stress. At approximately 5-5.5 months of age, MILON mice start to show signs of disease, followed by death shortly thereafter. The debut of overt disease in MILON mice coincides with onset of rapidly progressive neurodegeneration and massive cell death in hippocampus and neocortex. This profound neurodegenerative process is manifested as axonal degeneration, gliosis, and abundant TUNEL-positive nuclei. The MILON mouse model provides a novel and powerful tool for additional studies of the role for respiratory chain deficiency in neurodegeneration and aging.

Downregulation of Tfam and mtDNA copy number during mammalian spermatogenesis.

Mitochondrial transcription factor A (Tfam) is required for mtDNA maintenance, and mitochondrial Tfam protein levels directly affect mtDNA copy number. Previous studies have shown significant reduction of Tfam protein levels in mitochondria together with the appearance of abundant testis-specific Tfam mRNA isoforms as spermatogenesis proceeds in both mouse and man. Interestingly, an abundant testis-specific nuclear Tfam protein isoform of unknown function is found in the mouse, but not in humans. We have now characterized Tfam expression in rat testis to identify conserved features in mammalian spermatogenesis. The nuclear Tfam protein isoform is absent in the rat and is thus dispensable for mammalian spermatogenesis. Similar to mice and humans, we found expression of alternate Tfam transcripts, downregulation of mitochondrial Tfam protein levels, and downregulation of mtDNA copy number during rat spermatogenesis. These features are thus common to all mammals and may provide one of several mechanisms preventing paternal mtDNA transmission.